Respiratory organoids with externally attached ciliated epithelial cells and method for preparing the same

Abstract

The present disclosure relates to a method for preparing a respiratory organoid by proliferating and differentiating respiratory epithelial cells. Unlike the conventional respiratory organoids in which epithelial cells existing in the apical region of the respiratory organ are generated on the interior of the organoid, the respiratory organoid of the present disclosure is shown in a form in which ciliated structures, which are epithelial cells, are located in the outer layer of the organoid, secretory cells are present on the interior, and basal stem cells existing at the base of respiratory tissue are also present on the interior of the organoid. It has a form similar to an actual respiratory tissue, and it was observed that the epithelial cells exhibited normal function, such as motility of ciliated cells. Therefore, when an organoid is prepared using the preparation method of the present disclosure, a respiratory organoid that can well mimic actual tissue can be prepared.

1 . A method for preparing a respiratory organoid, in which ciliated epithelial cells are present on the exterior, comprising:
 (a) culturing respiratory cells using an expansion medium on a plate coated with an extracellular matrix material;   (b) obtaining the cultured cells of Step (a) and removing the expansion medium by centrifugation, thereby obtaining a cell pellet; and   (c) resuspending the cell pellet in a differentiation medium, and then inoculating and culturing a certain number of cells on a plate to be differentiated.   
     
     
         2 . The method of  claim 1 , wherein the respiratory organoid is a lung organoid. 
     
     
         3 . The method of  claim 1 , wherein the respiratory cell is a normal human bronchial basal epithelial cell (NHBE cell). 
     
     
         4 . The method of  claim 1 , wherein the extracellular matrix material of Step (a) is laminin, collagen I, or collagen IV. 
     
     
         5 . The method of  claim 4 , wherein the extracellular matrix material of Step (a) is laminin. 
     
     
         6 . The method of  claim 1 , wherein the expansion medium used in Step (a) comprises A8301, Y-27632, CHIR99021, and SB202190. 
     
     
         7 . The method of  claim 6 , wherein the expansion medium comprises a bronchial epithelial expansion medium (BEGM), 2 μM of A8301, 2 to 10 μM of Y-27632, 0.5 to 3 μM of CHIR99021, and 0.1 to 2 μM of SB202190. 
     
     
         8 . The method of  claim 1 , wherein the number of cells inoculated in Step (c) is 400 to 700. 
     
     
         9 . The method of  claim 1 , wherein Step (c) is performed for 15 to 30 days. 
     
     
         10 . The method of  claim 1 , wherein the respiratory organoid comprises basal stem cells on the interior of the organoid. 
     
     
         11 . A respiratory organoid prepared by the method of  claim 1 . 
     
     
         12 . The respiratory organoid of  claim 11 , wherein the respiratory organoid has a constitution in that ciliated cells are located on the exterior of the organoid, goblet cells, which are secretory cells, are located inner side thereof, and basal stem cells are located on the interior of the organoid. 
     
     
         13 . A method for evaluating the toxicity of a harmful substance, comprising:
 (a) treating a respiratory organoid prepared by the method of  claim 1  with a harmful substance;   (b) measuring the viability of cells in the respiratory organoid of the treatment group treated with the harmful substance in Step (a) and cells in the respiratory organoid of the control group which are not treated with the harmful substance; and   (c) determining the toxicity of the harmful substance by comparing the viability of cells measured in Step (b).